1. Field of the Invention
The present invention relates generally to the fields of medicine and immunology. In certain aspects, the field of the invention concerns immunotherapy. More particularly, it concerns the manufacture of clinical-grade polyclonal γδ T cells and therapeutic methods using such cells.
2. Description of Related Art
Human γδ T cells have both innate and adaptive qualities exhibiting a range of effector functions, including cytolysis upon cell contact (Bonneville et al., 2010). Recognition and subsequent killing of tumor target cells is achieved by heterodimers of γ and δ T-cell receptor (TCR) chains. The human TCR variable (V) region defines 14 unique Vγ alleles, 3 unique Vδ alleles (Vδ1, Vδ2, and Vδ3), and 5 Vδ alleles that share a common nomenclature with Vα alleles (Vδ4/Vα14, Vδ5/Vα29, Vδ6/Vα23, Vδ7/Vα36, and Vδ8/Vα38-2) (Lefranc, 2001). T cells expressing TCRα/TCRβ heterodimers compose approximately 95% of peripheral blood (PB) T cells and recognize peptides in the context of major histocompatibility complex (MHC) molecules (Turchinovich and Pennington, 2011). In contrast, TCRγδ ligands are recognized independent of MHC restriction but are infrequent (1%-5% of T cells) in PB (Bonneville et al., 2010; Kabelitz et al., 2007; Xu et al., 2011).
Human γδ T cells exhibit an inherent ability to lyse tumor cells and hold promise for immunotherapy. As such, many TCRγδ ligands are present on cancer cells, raising the possibility that an expansion approach that maintains a polyclonal repertoire of γδ TCRs has appeal for human application. Adoptive transfer of Vγ9Vδ2 T cells has yielded objective clinical responses for investigational treatment of cancer, but administration of non-Vγ9Vδ2 T cells has yet to be performed (Kondo et al., 2008; Lang et al., 2011; Nagamine et al., 2009; Nicol et al., 2011; Wilhelm et al., 2003). Long-term remissions of leukemia among recipients of haploidentical αβ T cell-depleted hematopoietic stem cell transplant (HSCT) correlated with increased engraftment frequency of donor-derived Vδ1 T cells (Godder et al., 2007; Lamb et al., 1999; Lamb et al., 1996; Lamb et al., 2001). No reports to date have described the therapeutic impact of Vδ1negVδ2neg T cells and this subset has not been directly compared to T cells expressing Vδ1 and Vδ2 TCRs. Thus, there are significant gaps in the knowledge and human application of non-Vγ9Vδ2 lineages.
Aminobisphosphonates, e.g., zoledronic acid (Zol), have been used to propagate the Vγ9Vδ2 subset of γδ T cells for clinical use (Stresing et al., 2007; Thompson et al., 2010). Other γδ T cell lineages are not propagated by aminobisphosphonates. Nonetheless, clinical trials that have used Vγ9Vδ2 γδ T cells as cancer immunotherapies have shown some objective responses but were not curative as a single therapy (Nicol et al., 2011; Wilhelm et al., 2003). Plate-bound antibodies and cytokine cocktails have also been used to propagate a more diverse set of γδ T cells, but (i) they did not achieve consistent Vδ1 and Vδ1negVδ2neg frequencies, (ii) the absolute numbers of γδ T cells were not clinically-relevant (<108 cells), (iii) they did not comprehensively analyze Vγ frequencies, and (iv) they are not as directly translatable to the clinic as these reagents are not all available at good manufacturing practices (GMP) quality (Dokouhaki et al., 2010; Kang et al., 2009; Lopez et al., 2000). Therefore, clinically-relevant methods of expanding γδ T cells ex vivo, and the cells produced thereby, are greatly needed.